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RUMINICLOSTRIDIUM MEDIUM
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Selected strain: Clostridium polysaccharolyticum DSM 1801
This strain uses the standard medium without modifications.
Growth conditions: anaerobic
37 °C
Equipment needed:
Autoclave
Centrifuge
Filter
Gassing station
Hungate tubes
Final gas composition:
100 % CO2
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Final pH:
6.8
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.50 | g | ||
|
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NaCl | 1.00 | g | ||
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(NH4)2SO4 | 0.50 | g | ||
|
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MgSO4 x 7 H2O | 0.10 | g | ||
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CaCl2 x 2 H2O | 0.10 | g | ||
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K2HPO4 | 0.50 | g | ||
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Clarified rumen fluid | 300.00 | ml | ||
|
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Sludge fluid
, alternative
optional |
300.00 | ml | ||
|
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
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Na2CO3 | 2.50 | g | ||
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Cellobiose | 5.00 | g | ||
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L-Cysteine HCl x H2O | 0.25 | g | ||
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Na2S x 9 H2O | 0.25 | g | ||
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Distilled water | 700.00 | ml | ||
| 1 Dissolve ingredients (except carbonate, cellobiose and reducing agents), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Use either clarified rumen fluid or sludge fluid as supplement. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add cellobiose, sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Cellobiose has to be sterilized by filtration. Adjust pH of complete medium to 6.8, if necessary. | |||||
Sludge fluid #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
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Yeast extract | 4 | g | ||
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Sludge | 1000 | ml | ||
| 1 Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark. | |||||
Clarified rumen fluid #
1000
| 1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C. |